title: A clinically validated diagnostic second-generation sequencing assay for detection of hereditary BRCA1 and BRCA2 mutations.
abstract: Individuals who inherit mutations in BRCA1 or BRCA2 are predisposed to breast and ovarian cancers. However, identifying mutations in these large genes by conventional dideoxy sequencing in a clinical testing laboratory is both time consuming and costly, and similar challenges exist for other large genes, or sets of genes, with relevance in the clinical setting. Second-generation sequencing technologies have the potential to improve the efficiency and throughput of clinical diagnostic sequencing, once clinically validated methods become available. We have developed a method for detection of variants based on automated small-amplicon PCR followed by sample pooling and sequencing with a second-generation instrument. To demonstrate the suitability of this method for clinical diagnostic sequencing, we analyzed the coding exons and the intron-exon boundaries of BRCA1 and BRCA2 in 91 hereditary breast cancer patient samples. Our method generated high-quality sequence coverage across all targeted regions, with median coverage greater than 4000-fold for each sample in pools of 24. Sensitive and specific automated variant detection, without false-positive or false-negative results, was accomplished with a standard software pipeline using bwa for sequence alignment and samtools for variant detection. We experimentally derived a minimum threshold of 100-fold sequence depth for confident variant detection. The results demonstrate that this method is suitable for sensitive, automatable, high-throughput sequence variant detection in the clinical laboratory.
authors.affiliation: Cancer Genetics Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Ian E
authors.initials: IE
authors.lastname: Bosdet
authors.forename: T Roderick
authors.initials: TR
authors.lastname: Docking
authors.forename: Yaron S
authors.initials: YS
authors.lastname: Butterfield
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Mungall
authors.forename: Thomas
authors.initials: T
authors.lastname: Zeng
authors.forename: Robin J
authors.initials: RJ
authors.lastname: Coope
authors.forename: Erika
authors.initials: E
authors.lastname: Yorida
authors.forename: Katie
authors.initials: K
authors.lastname: Chow
authors.forename: Miruna
authors.initials: M
authors.lastname: Bala
authors.forename: Sean S
authors.initials: SS
authors.lastname: Young
authors.forename: Martin
authors.initials: M
authors.lastname: Hirst
authors.forename: Inanc
authors.initials: I
authors.lastname: Birol
authors.forename: Richard A
authors.initials: RA
authors.lastname: Moore
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Jones
authors.forename: Marco A
authors.initials: MA
authors.lastname: Marra
authors.forename: Rob
authors.initials: R
authors.lastname: Holt
authors.forename: Aly
authors.initials: A
authors.lastname: Karsan
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D005787:Gene Frequency; D015723:Gene Library; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D061325:Hereditary Breast and Ovarian Cancer Syndrome / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D011446:Prospective Studies; D012680:Sensitivity and Specificity